Coagulation and fibrinolysis are two apparently independent processes which together constitute the haemostatic system of the blood. This system is responsible for, on one side, keeping the free-flow of blood through the blood vessels and on the other side preventing excessive blood loss following a wound. The enzyme thrombin [EC 3.4.21.5] plays a pivotal role in both situations and, indeed is the most important link between both processes. Thrombin production is a complex event that includes generation of active thrombin from precursors as well as inactivation of the formed thrombin by physiological inhibitors. Further, thrombin itself promotes its own formation and inactivation. It has become clear that measuring the progress of thrombin activity in time (thrombin generation) results in a global appraisal of the haemostatic system (1, 2).
The necessity to measure the function of the haemostatic system has not escaped the attention of the medical profession over the last century. Since the 50ies, it has been recognized that measuring the time course of thrombin in clotting blood gave the best estimate of the functionality of the haemostatic system, as well as of the propensity towards thrombotic or bleeding states. From the eighties on, fundamental work from HC Hemker's laboratory in Maastricht resulted in the development of a computerized, automatic method that allows estimation of the thrombin generation parameters with relatively high output, from Platelet-free (PPP) as well as Platelet-rich plasma (PRP) [EP-B2-0420332 and WO 03/093831A1]. Following the basic principles of Hemker's development, other groups have implemented alternative methods to measure thrombin generation in plasma which, due to the lack of appropriate calibration [WO 03/093831 A1], give variable and imprecise estimates of the thrombin generation parameters [[1,2]].
Up to this date, all the existing methods for measuring thrombin generation in plasma need an additional, non-automatic step, previous to the actual measurement. That is, in order to obtain PPP or PRP the sample of whole blood have to be subjected to centrifugation in order to separate the cellular components of the blood from the plasma. Besides the obvious extra manipulation of the sample, this step, by depleting the sample from cells, will not allow the estimation of their contribution to the generation of thrombin.
The present invention provides a method by which triggering of thrombin generation will be done in a sample of whole blood, while the actual measurement of the progress of thrombin activity in time will be followed in plasma, in a single step, without the need for a previous separation of the blood cells.